Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Non-BRCA1/BRCA2 high-risk familial breast cancers are not associated with a high prevalence of BRCAness.

BACKGROUND: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown. METHODS: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers. RESULTS: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent. CONCLUSIONS: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells.

Heterogeneity and tumor evolution reflected in liquid biopsy in metastatic breast cancer patients: a review.

Breast cancer is a spatially and temporally dynamic disease in which differently evolving genetic clones are responsible for progression and clinical outcome. We review tumor heterogeneity and clonal evolution from studies comparing primary tumors and metastasis and discuss plasma circulating tumor DNA as a powerful real-time approach for monitoring the clonal landscape of breast cancer during treatment and recurrence. We found only a few early studies exploring clonal evolution and heterogeneity through analysis of multiregional tissue biopsies of different progression steps in comparison with circulating tumor DNA (ctDNA) from blood plasma. The model of linear progression seemed to be more often reported than the model of parallel progression. The results show complex routes to metastasis, however, and plasma most often reflected metastasis more than primary tumor. The described patterns of evolution and the polyclonal nature of breast cancer have clinical consequences and should be considered during patient diagnosis and treatment selection. Current studies focusing on the relevance of clonal evolution in the clinical setting illustrate the role of liquid biopsy as a noninvasive biomarker for monitoring clonal progression and response to treatment. In the clinical setting, circulating tumor DNA may be an ideal support for tumor biopsies to characterize the genetic landscape of the metastatic disease and to improve longitudinal monitoring of disease dynamics and treatment effectiveness through detection of residual tumor after resection, relapse, or metastasis within a particular patient.

Whole blood transcriptional profiling reveals highly deregulated atherosclerosis genes in Philadelphia-chromosome negative myeloproliferative neoplasms.

BACKGROUND: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPN but also potentially having an impact upon the development of accelerated (premature) atherosclerosis. OBJECTIVES: Since chronic inflammation, atherosclerosis, and atherothrombosis are prevalent in MPNs and we have previously shown oxidative stress genes to be markedly upregulated in MPNs, we hypothesized that genes linked to development of atherosclerosis might be highly deregulated as well. METHODS: Using whole blood gene expression profiling in patients with essential thrombocythemia (ET; n = 19), polycythemia vera (PV; n = 41), or primary myelofibrosis (PMF; n = 9), we herein for the first time report aberrant expression of several atherosclerosis genes. RESULTS: Of 84 atherosclerosis genes, 45, 56, and 46 genes were deregulated in patients with ET, PV, or PMF, respectively. Furthermore, BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated and BCL2 progressively significantly downregulated from ET over PV to PMF (all FDR <0.05). CONCLUSIONS: We have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of leukocytes, platelets, and endothelial cells being deeply involved in the atherosclerotic process.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Tumour-infiltrating CD4-, CD8- and FOXP3-positive immune cells as predictive markers of mortality in BRCA1- and BRCA2-associated breast cancer.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse. METHODS: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival. RESULTS: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers. CONCLUSIONS: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients.

Epithelial ovarian cancer and the use of circulating tumor DNA: A systematic review.

OBJECTIVE: One way to improve the survival rate of epithelial Ovarian Cancer (EOC) is by identifying effective biomarkers useful at different stages and time points of the disease. A potential biomarker is circulating tumor DNA (ctDNA) in plasma or serum. In this systematic review, we provide an overview of applications of ctDNA in EOC to discuss the direction of future research in this field. METHODS: We performed a systematic search in Pubmed, Embase, and Scopus to identify relevant clinical studies eligible for inclusion. Furthermore, the references in the identified studies and relevant reviews were assessed to identify additional studies. The PRISMA guideline was employed to perform the systematic review, and data from the studies were extracted using piloted data extraction forms. RESULTS: A total of 36 observational studies were included. The concordance between tumor and ctDNA was assessed in 19 studies, early diagnosis in 1, diagnosis in 23, monitoring of treatment response in 7, detection of reversion mutations in 3, prognosis in 9, but no studies assessed early detection of recurrence. Data from the studies were reported descriptively. The studies had a large variation in the methods used for ctDNA analysis and limited sample sizes of 10-126 patients. Overall, the studies show that ctDNA is a potential biomarker for EOC useful in several settings during assessment and treatment of these patients. CONCLUSIONS: Although the identified studies are limited in number and their methods for ctDNA analysis vary, it is clear that ctDNA as a biomarker for EOC is promising for several applications in diagnostics, monitoring of treatment response, and prognostics. However, more studies are needed to establish the ideal methods and settings for the clinical use of ctDNA in EOC.

Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study.

We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission was 0.85 and 0.75 for patients with polycythemia vera and myelofibrosis, respectively. The Myeloproliferative Neoplasm Symptom Assessment Form total symptom score decreased from 22 [95% confidence interval (95% CI):, 16-29] at baseline to 15 (95% CI: 10-22) after 2 years. The median JAK2 V617F allele burden decreased from 47% (95% CI: 33-61%) to 12% (95% CI: 6-22%), and 41% of patients achieved a molecular response. The drop-out rate was 6% among patients with polycythemia vera and 32% among those with myelofibrosis. Of 36 patients previously intolerant of PEG-IFNα2, 31 (86%) completed the study, and 24 (67%) of these received PEG-IFNα2 throughout the study. In conclusion, combination treatment improved cell counts, reduced bone marrow cellularity and fibrosis, decreased JAK2 V617F burden, and reduced symptom burden with acceptable toxicity in several patients with polycythemia vera or myelofibrosis. #EudraCT2013-003295-12.

The BRCA1 c. 5096G>A p.Arg1699Gln (R1699Q) intermediate risk variant: breast and ovarian cancer risk estimation and recommendations for clinical management from the ENIGMA consortium.

BACKGROUND: We previously showed that the BRCA1 variant c.5096G>A p.Arg1699Gln (R1699Q) was associated with an intermediate risk of breast cancer (BC) and ovarian cancer (OC). This study aimed to assess these cancer risks for R1699Q carriers in a larger cohort, including follow-up of previously studied families, to further define cancer risks and to propose adjusted clinical management of female BRCA1*R1699Q carriers. METHODS: Data were collected from 129 BRCA1*R1699Q families ascertained internationally by ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium members. A modified segregation analysis was used to calculate BC and OC risks. Relative risks were calculated under both monogenic model and major gene plus polygenic model assumptions. RESULTS: In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83). CONCLUSION: Our results confirm that BRCA1*R1699Q confers an intermediate risk for BC and OC. Breast surveillance for female carriers based on mammogram annually from age 40 is advised. Bilateral salpingo-oophorectomy should be considered based on family history.

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.

Myeloproliferative Neoplasms in Danish Twins.

OBJECTIVE: Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases characterized by clonal hyperproliferation of immature and mature cells of the myeloid lineage. Genetic differences have been proposed to play a role in the development of MPNs. Monozygotic twin pairs with MPNs have been reported in a few case reports, but the MPN concordance pattern in twins remains unknown. METHOD: All twin pairs born in the period 1900-2010 were identified in the nationwide Danish Twin Registry. Only pairs with both twins alive on January 1, 1977, and those born thereafter were included to allow identification in the Danish National Patient Registry. RESULTS: A total of 158 twin pairs were registered with an MPN diagnosis: 36 monozygotic, 104 dizygotic, and 18 pairs with unknown zygosity. MPNs were diagnosed in both twins in 4 pairs. The probandwise concordance rates for monozygotic twin pairs were higher than for dizygotic twin pairs (15 vs. 0%; p = 0.016). CONCLUSION: An estimated concordance rate of 15% (95% CI 0.059-0.31) is modest, but given the rarity of MPNs this finding is clinically relevant and provides further support for the role of genetic predisposition in the development of MPNs.

The gene expression and immunohistochemical time-course of diphenylcyclopropenone-induced contact allergy in healthy humans following repeated epicutaneous challenges.

The gene expression time-course of repeated challenge of contact allergy (CA) remains largely unknown. Therefore, using diphenylcyclopropenone (DPCP) as model allergen in healthy humans we set out to examine: (i) the monotonous and complex gene expression time-course trajectories following repeated DPCP challenges to find the predominant gene expression pattern, (ii) the time-course of cell infiltration following repeated DPCP challenges and (iii) the transcriptome of a repeated CA exposure model. We obtained punch biopsies from control and DPCP-exposed skin from ten DPCP sensitized individuals at 5-6 monthly elicitation challenges. Biopsies were used for microarray gene expression profiling, histopathology and immunohistochemical staining. Validation of microarray data by qRT-PCR was performed on 15 selected genes. Early gene expression time points were also validated in an independent data set. An increasing and decreasing trend in gene expression followed by a plateau was predominantly observed during repeated DPCP challenges. Immune responses reached a plateau after two challenges histopathologically, immunohistochemically and in the time-course gene expression analysis. Transcriptional responses over time revealed a Th1/Th17 polarization as three upstream regulators (IFN-γ, IL-1 and IL-17) activated most of the top upregulated genes. Of the latter genes, 9 of 10 were the same throughout the time course. Excellent correlations between array and PCR data were observed. The transcriptional responses to DPCP over time followed a monotonous pattern. This response pattern confirms and supports the newly reported clinical time-course observations in de novo-sensitized individuals showing a plateau response, and thus, there is concordance between clinical response, histopathology, immunohistochemistry and microarray gene expression in volunteers de novo-sensitized to DPCP.

Myelin-specific T cells induce interleukin-1beta expression in lesion-reactive microglial-like cells in zones of axonal degeneration.

Infiltration of myelin-specific T cells into the central nervous system induces the expression of proinflammatory cytokines in patients with multiple sclerosis (MS). We have previously shown that myelin-specific T cells are recruited into zones of axonal degeneration, where they stimulate lesion-reactive microglia. To gain mechanistic insight, we used RNA microarray analysis to compare the transcript profile in hippocampi from perforant pathway axonal-lesioned mice with and without adoptively transferred myelin-specific T cells 2 days postlesion, when microglia are clearly lesion reactive. Pathway analysis revealed that, among the 1,447 differently expressed transcripts, the interleukin (IL)-1 pathway including all IL-1 receptor ligands was upregulated in the presence of myelin-specific T cells. Quantitative polymerase chain reaction showed increased mRNA levels of IL-1ß, IL-1α, and IL-1 receptor antagonist in the T-cell-infiltrated hippocampi from axonal-lesioned mice. In situ hybridization and immunohistochemistry showed a T-cell-enhanced lesion-specific expression of IL-1ß mRNA and protein, respectively, and induction of the apoptosis-associated speck-like protein, ASC, in CD11b(+) cells. Double in situ hybridization showed colocalization of IL-1ß mRNA in a subset of CD11b mRNA(+) cells, of which many were part of cellular doublets or clusters, characteristic of proliferating, lesion-reactive microglia. Double-immunofluorescence showed a T-cell-enhanced colocalization of IL-1ß to CD11b(+) cells, including lesion-reactive CD11b(+) ramified microglia. These results suggest that myelin-specific T cells stimulate lesion-reactive microglial-like cells to produce IL-1ß. These findings are relevant to understand the consequences of T-cell infiltration in white and gray matter lesions in patients with MS.

Migrating glioma cells express stem cell markers and give rise to new tumors upon xenografting.

Glioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy, recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell-like phenotype is currently lacking. In the present study, the aim was to characterize the phenotype of migrating tumor cells using a novel migration assay based on serum-free stem cell medium and patient-derived spheroid cultures. The results showed pronounced migration of five different GBM spheroid cultures, but not of the commercial cell line U87MG. An in vitro limiting dilution assay showed preserved but reduced spheroid formation capacity of migrating cells. Orthotopic xenografting in mice showed preserved but reduced tumorigenic capacity. Profiling of mRNAs revealed no significant deregulation of 16 predefined CSC-related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since CSCs are reported to be highly resistant to therapy, these results emphasize that the CSC phenotype should be taken into consideration in future treatment of GBMs.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures.

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

Chemotherapy Modulates Intestinal Immune Gene Expression Including Surfactant Protein-D and Deleted in Malignant Brain Tumors 1 in Piglets.

BACKGROUND: Information about chemotherapy-induced intestinal gene expression may provide insight into the mechanisms underlying gut toxicity and help identify biomarkers and targets for intervention. METHODS: We analyzed jejunal tissue from piglets subjected to two different, clinically relevant chemotherapy regimens: (1) busulfan plus cyclophosphamide (BUCY) and (2) doxorubicin (DOX). RESULTS: Gene expression analysis identified 1,328 differentially expressed genes in the BUCY piglets and 594 in the DOX piglets, compared to controls. Similar changes in expression were found for 137 genes across the BUCY and DOX piglets. Selected genes of potential biological significance with a similar change in expression across the treatments were controlled by real-time polymerase chain reaction. Key innate defense molecules, including surfactant protein-D and deleted in malignant brain tumors 1, were among the upregulated genes for both treatments. CONCLUSION: In the developing intestine, chemotherapy increases the expression of genes related to innate immune functions involved in surveillance, protection, and homeostasis of mucosal surfaces.

Molecular Concordance Between Primary Breast Cancer and Matched Metastases.

Clinical management of breast cancer is increasingly personalized and based on molecular profiling. Often, primary tumors are used as proxies for systemic disease at the time of recurrence. However, recent studies have revealed substantial discordances between primary tumors and metastases, both with respect to traditional clinical treatment targets and on the genomic and transcriptomic level. With the increasing use of molecularly targeted therapy, discordance of actionable molecular targets between primary tumors and recurrences can result in nonoptimal treatment or unnecessary side effects. The purpose of this review is to illuminate the extent of cancer genome evolution through disease progression and the degree of molecular concordance between primary breast cancers and matched metastases. We present an overview of the most prominent studies investigating the expression of endocrine receptors, transcriptomics, and genome aberrations in primary tumors and metastases. In conclusion, biopsy of metastatic lesions at recurrence of breast cancer is encouraged to provide optimal treatment of the disease. Furthermore, molecular profiling of metastatic tissue provides invaluable mechanistic insight into the biology underlying metastatic progression and has the potential to identify novel, potentially druggable, drivers of progression.

Long non-coding RNA expression profiles predict metastasis in lymph node-negative breast cancer independently of traditional prognostic markers.

INTRODUCTION: Patients with clinically and pathologically similar breast tumors often have very different outcomes and treatment responses. Current prognostic markers allocate the majority of breast cancer patients to the high-risk group, yielding high sensitivities in expense of specificities below 20%, leading to considerable overtreatment, especially in lymph node-negative patients. Seventy percent would be cured by surgery and radiotherapy alone in this group. Thus, precise and early indicators of metastasis are highly desirable to reduce overtreatment. Previous prognostic RNA-profiling studies have only focused on the protein-coding part of the genome, however the human genome contains thousands of long non-coding RNAs (lncRNAs) and this unexplored field possesses large potential for identification of novel prognostic markers. METHODS: We evaluated lncRNA microarray data from 164 primary breast tumors from adjuvant naïve patients with a mean follow-up of 18 years. Eighty two patients who developed detectable distant metastasis were compared to 82 patients where no metastases were diagnosed. For validation, we determined the prognostic value of the lncRNA profiles by comparing the ability of the profiles to predict metastasis in two additional, previously-published, cohorts. RESULTS: We showed that lncRNA profiles could distinguish metastatic patients from non-metastatic patients with sensitivities above 90% and specificities of 64-65%. Furthermore; classifications were independent of traditional prognostic markers and time to metastasis. CONCLUSIONS: To our knowledge, this is the first study investigating the prognostic potential of lncRNA profiles. Our study suggest that lncRNA profiles provide additional prognostic information and may contribute to the identification of early breast cancer patients eligible for adjuvant therapy, as well as early breast cancer patients that could avoid unnecessary systemic adjuvant therapy. This study emphasizes the potential role of lncRNAs in breast cancer prognosis.

Ensemble-based classification using microRNA expression identifies a breast cancer patient subgroup with an ultralow long-term risk of metastases.

BACKGROUND: Current clinical markers overestimate the recurrence risk in many lymph node negative (LNN) breast cancer (BC) patients such that a majority of these low-risk patients unnecessarily receive systemic treatments. We tested if differential microRNA expression in primary tumors allows reliable identification of indolent LNN BC patients to provide an improved classification tool for overtreatment reduction in this patient group. METHODS: We collected freshly frozen primary tumors of 80 LNN BC patients with recurrence and 80 recurrence-free patients (mean follow-up: 20.9 years). The study comprises solely systemically untreated patients to exclude that administered treatments confound the metastasis status. Samples were pairwise matched for clinical-pathological characteristics to minimize dependence of current markers. Patients were classified into risk-subgroups according to the differential microRNA expression of their tumors via classification model building with cross-validation using seven classification methods and a voting scheme. The methodology was validated using available data of two independent cohorts (n = 123, n = 339). RESULTS: Of the 80 indolent patients (who would all likely receive systemic treatments today) our ultralow-risk classifier correctly identified 37 while keeping a sensitivity of 100% in the recurrence group. Multivariable logistic regression analysis confirmed independence of voting results from current clinical markers. Application of the method in two validation cohorts confirmed successful classification of ultralow-risk BC patients with significantly prolonged recurrence-free survival. CONCLUSION: Profiles of differential microRNAs expression can identify LNN BC patients who could spare systemic treatments demanded by currently applied classifications. However, further validation studies are required for clinical implementation of the applied methodology.

Common alleles at 6q25.1 and 1p11.2 are associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers.

Two single nucleotide polymorphisms (SNPs) at 6q25.1, near the ESR1 gene, have been implicated in the susceptibility to breast cancer for Asian (rs2046210) and European women (rs9397435). A genome-wide association study in Europeans identified two further breast cancer susceptibility variants: rs11249433 at 1p11.2 and rs999737 in RAD51L1 at 14q24.1. Although previously identified breast cancer susceptibility variants have been shown to be associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers, the involvement of these SNPs to breast cancer susceptibility in mutation carriers is currently unknown. To address this, we genotyped these SNPs in BRCA1 and BRCA2 mutation carriers from 42 studies from the Consortium of Investigators of Modifiers of BRCA1/2. In the analysis of 14 123 BRCA1 and 8053 BRCA2 mutation carriers of European ancestry, the 6q25.1 SNPs (r(2) = 0.14) were independently associated with the risk of breast cancer for BRCA1 mutation carriers [hazard ratio (HR) = 1.17, 95% confidence interval (CI): 1.11-1.23, P-trend = 4.5 × 10(-9) for rs2046210; HR = 1.28, 95% CI: 1.18-1.40, P-trend = 1.3 × 10(-8) for rs9397435], but only rs9397435 was associated with the risk for BRCA2 carriers (HR = 1.14, 95% CI: 1.01-1.28, P-trend = 0.031). SNP rs11249433 (1p11.2) was associated with the risk of breast cancer for BRCA2 mutation carriers (HR = 1.09, 95% CI: 1.02-1.17, P-trend = 0.015), but was not associated with breast cancer risk for BRCA1 mutation carriers (HR = 0.97, 95% CI: 0.92-1.02, P-trend = 0.20). SNP rs999737 (RAD51L1) was not associated with breast cancer risk for either BRCA1 or BRCA2 mutation carriers (P-trend = 0.27 and 0.30, respectively). The identification of SNPs at 6q25.1 associated with breast cancer risk for BRCA1 mutation carriers will lead to a better understanding of the biology of tumour development in these women.